Treatment of metastatic tumors is a major health challenge. Recently, antibody-based therapies have been developed that are more specific and have fewer side effects compared with conventional chemotherapy. However, the potency of most antibody therapeutics is limited by their inadequate ability to kill tumor cells. Consequently, there is an urgent, unmet need to develop therapeutics that combine the specificity of antibodies for tumor tissues with a potent cytotoxic function. The development of tumor-targeted toxins has yielded promising results and led to one approved product, Ontak (Denileukin). Most molecules however are immunogenic and aggregation prone, have limited stability and require complex manufacturing routes. To achieve clinical and commercial success it is critical for candidates to meet following criteria: 1) high potency;2) low systemic toxicity;3) low immunogenicity;4) high protein stability, lack of aggregation;5) robust manufacturing. This proposal aims to develop tumor-targeted toxins by combining three elements that confer significant advantages over current approaches: microproteins for tumor binding/internalization;RNAse for cell killing;rPEG to optimize PK properties. In the successful Phase I of this project, we developed tumor-specific microproteins with the following properties: 1) efficient production in E. coli;2) efficient phage display that enables rapid specificity optimization;3) effective internalization of toxic payloads;4) excellent serum stability. In a separate phase I SBIR project, we developed rPEGs, hydrophilic protein sequences that mimic the properties of chemical polyethylene glycol (PEG) but can be directly fused to other proteins. rPEGs optimize the pharmacokinetics of a product, reduce product immunogenicity, and greatly reduce protein aggregation. Our Phase II goal is to optimize the specificity of our lead microproteins to achieve a >1000x ration of tumor/normal affinity. Subsequently, we will fuse these optimized microproteins to RNAse as toxic payload and rPEG to optimize PK, PD and protein manufacturing. The resulting fusion proteins will be thoroughly evaluated for in vitro and in vivo performance. In addition, we will develop an effective manufacturing process that can be transferred with minor modifications to a GMP manufacturer. We aim to generate two lead molecules that will be ready to enter preclinical followed by clinical development. In addition we will generate microprotein-rPEG fusions with defined conjugation sites that will be uniquely suitable for the chemical conjugation of toxic payloads. PUBLIC HEALTH RELEVANCE: The development of tumor-targeted toxins have yielded promising results and led to one approved product, Denileukin. However, existing molecules have significant limitations especially immunogenicity and complex manufacturing requirements. This project will use tumor-specific microproteins to address these limitations and develop targeted toxins with the following characteristics: 1) high potency;2) low systemic toxicity;3) low immunogenicity to allow repeat dosing;4) good protein stability;5) lack of aggregation;6) robust manufacturing process.